A variety of techniques are known for the introduction of foreign substances, particularly DNA, into the interiors of living cells without killing those same cells. One class of techniques involves manipulating the cell membrane to make it permeable to DNA molecules. For instance, in bacteria, yeasts and protoplasts of higher plant cells, treatments with chemicals or heat can be used to make the cell membranes "leaky," thereby permitting a desired gene or genes (often integrated in small loops of DNA called plasmids) to be taken up into the cell.
In another class of techniques, DNA is physically injected into living cells, particularly ova, of various animal species. DNA can also be taken up by electrically stimulated cells in a process known as electroporation. In another procedure, the cell is made permeable using a precision laser to burn holes into the cell membrane.
Yet another type of gene introduction can be accomplished using naturally occurring processes. Gene transfer is accomplished by infecting certain susceptible dicotyledonous plant species with a particular bacterial species of the genus Agrobacterium. The bacterium possesses plasmids as a part of its normal complement of DNA. Upon infection, the bacterium is capable of transferring portions of these plasmids to the cells of the plant it infects. Thus, it is possible to accomplish the introduction of specifically desired genes by engineering Agrobacterium species with recombinant plasmids possessing the desired gene and subsequently infecting a susceptible plant with the engineered bacterium.
Most recently, another type of gene introduction method has been described. Termed "microprojectile bombardment" or "biolistics," this technique involves coating tiny metal spheres with desired DNA, and then accelerating these projectiles into tissues. Acceleration is generally achieved by shooting the microprojectiles from a gun aimed at the target tissue. Once inside the cell, the foreign DNA detaches from the metal sphere and, in certain of the bombarded cells, is incorporated into the host cell's DNA. See, e.g., Sanford, J. C., "The biolistic process," Trends in Biotechnology, 6:299-302 (1988); Klein, T. et al. "Stable genetic transformation of intact Nicotiana cells by the particle bombardment process," Proc. Nat. Acad. Sci. USA 85:8502-8505 (1988); E.P. Application, Ser. No. 87310612.4; E.P. Application, Ser. No. 88306613.6; and E.P. Application, Ser. No. 88402481.1.
Unfortunately, each of the above methods has distinct disadvantages. The methods may be taxonomically limited, such as with Agrobacterium-based methods. Membrane permeability methods have limited application with organisms presenting cell walls. Electroporation methods achieve only a very low efficiency of stable transformation. Several methods, such as microinjection and laser-mediated cell membrane manipulation are extremely tedious, time consuming and require highly specialized equipment.
Although the biolistics approach avoids some of these drawbacks, the design and composition of the microprojectiles presents disadvantages unique to the method. For instance, metal microprojectiles can easily clump into larger aggregates that, upon impact, can cause significant cell damage. The type of substances that can be introduced into the cell is limited to negatively charged molecules such as DNA and RNA. The amount of DNA or RNA carried with each microprojectile cannot be easily controlled or measured. The long-term chemical and physical effects of the metal microprojectiles inside the cell are unknown. Finally, the use of metallic agents requires considerable preparation time, thus affecting the economic efficiency of the method.